Removal of leaked affinity purification ligand

ABSTRACT

The invention provides for the removal of a large fraction of contaminants from protein preparations while maintaining a high level of recovery using tentacle anion exchange matrix chromatography medium. Using the methods of the invention, leached affinity chromatography contaminants can be removed from recombinant protein preparations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 17/517,190, filed Nov. 2, 2021, which is a continuation of U.S.application Ser. No. 16/042,965, filed Jul. 23, 2018, which is acontinuation application U.S. application Ser. No. 14/775,992, filedSep. 14, 2015, which is a 371 of PCT/US2014/023682, filed Mar. 11, 2014,which claims priority to the benefit of U.S. Provisional Application No.61/785,038, filed Mar. 14, 2013. The above-identified applications areeach hereby incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The invention is in the field of removal of contaminants from proteinpreparations. In one aspect, the invention relates to the removal ofleached affinity chromatography contaminants from a protein preparation.

BACKGROUND OF THE INVENTION

Affinity chromatography is a powerful tool for purification of proteinssuch as antibodies and Fc-fusion proteins. However, if the proteins aremanufactured for therapeutic use, the presence of other proteins,including a protein used as part of an affinity adsorbent, which canleach into a sample during affinity chromatography, is of concern. Inaddition, other protein contaminants may also be present in a sample,such as, for example, proteins derived from host cells that produce theprotein being purified.

Proteins A and G are often employed to purify antibodies by affinitychromatography. Ford et al. (2001), J. Chromatogr. B 754: 427-435. Theseproteins are useful because they bind to a constant (Fc) portion of manydifferent antibodies. Recombinant fusion proteins including an Fcportion of an IgG antibody can be purified using similar methods. Inmost protein A and protein G affinity chromatography operations, smallamounts of the protein A or G ligand leach from the affinity column andend up in the eluate as either free ligand or ligand in complex with thetarget protein. If the antibody or fusion protein will be used as atherapeutic, the leached ligand and ligand/protein complexes must beremoved.

Manufacturers of chromatography resins recommend using ion exchangechromatography to remove residual contaminants such as protein A. See,e.g., “Process Scale Antibody Purification” Application Note 11-0011-64AA, 2004-11 (GE Healthcare). Kelley et al. reported that cation exchangechromatography is more typically used in a bind and elute mode, whereimpurities less basic than the product are removed in the load and wash,and more basic impurities are separated from the product during elution.Kelley et al., 2008, Biotechnol. and Bioeng., vol. 100: 950-963.According to the same authors, anion exchange chromatography is usuallyoperated in a flow through mode, since polyanions such as endotoxin andnucleic acids can be bound to the column under conditions that allow thedesired product to flow through. Id. at 950. Anion exchangechromatography in a flow through mode can also typically be used toremove host cell proteins and protein A. Id. at 961.

Hydroxyapatite chromatography is surprisingly effective at removingleached protein A during process scale protein production. U.S. Pat. No.7,476,722 describes that a hydroxyapatite column can be operated in flowthrough mode while maintaining over 90% recovery of the target proteinand reduction in contaminating Protein A of from 5.3 to 5.4 fold.However, in some situations, hydroxyapatite may not be convenient to usebecause of mechanical instability and/or low reusability, or may notsufficiently remove all of the leached protein A. Thus, there is a needin the art for alternative ways of removing leached protein A, while atthe same time maximizing recovery of protein and thereby controlling thecosts of recombinant protein production.

SUMMARY OF THE INVENTION

Although affinity chromatography is a highly effective technique forisolating proteins, one drawback is that the affinity ligand maycontaminate the resulting sample of recombinant protein by leaching fromthe affinity chromatography medium. Because affinity ligands are chosenfor their ability to associate with the recombinant protein, it can bechallenging to remove them from a final preparation without also losingthe recombinant protein. The invention provides an effective, gentle,and easily scalable way of accomplishing this goal using tentacle anionexchange matrix chromatography medium. While not wishing to be bound toany particular mechanism, it is thought that the tentacle aspect of theanion exchange matrix chromatography medium gives the methods of theinvention unexpectedly better results than that observed with any othertype of anion exchange resin, or indeed most resins, in removingaffinity ligand without significant losses in recovery of therecombinant protein.

Accordingly, the method provides, in one aspect, a method for purifyinga recombinant protein from a sample containing the recombinant proteinand a second protein that binds to the protein, comprising subjectingthe sample to a tentacle anion exchange matrix chromatography mediumunder conditions whereby the recombinant protein binds to the tentacleanion exchange matrix chromatography medium, followed by eluting therecombinant protein bound to the chromatography medium in an eluant,whereby at least 85% of the recombinant protein is recovered in theeluant and at least 75% of the second protein is removed from theeluant. In one aspect, the tentacle anion exchange matrix chromatographymedium contains a strong anion functional group. Such a strong anionfunctional group can be trimethyl-ammoniumethyl (TMAE). The resinsubstrate of the tentacle anion exchange matrix chromatography mediumcan be a methacrylate polymeric resin or a polyvinylstyrene polymericresin. In one aspect of the above embodiments, the chromatography mediumis Fractogel® EMD TMAE HiCap.

In all of the above embodiments, the methods of the invention can beused to purify recombinant protein containing a C_(H)2/C_(H)3 region ofan antibody. Such proteins can be purified on affinity columns such as aProtein A or Protein G chromatography resin. Accordingly, the methods ofthe invention can be used when the second protein is Protein A orProtein G. In any one of the above embodiments, the sample can beobtained from affinity purification of the protein. Such an affinitypurification can be over a Protein A chromatography medium.

In any of the above aspects of the invention, the recombinant proteincan be an antibody or an Fc fusion protein. In one embodiment of theabove aspects, the recombinant protein is a tumor necrosis factorreceptor Fc fusion protein, such as, for example, etanercept.

Optionally, in any of the above aspects of the invention, after therecombinant protein is bound to the tentacle anion exchange matrixchromatography medium and before the recombinant protein is eluted, thetentacle anion exchange matrix chromatography medium can subjected to awash step. As another option in any of the above aspects of theinvention, the recombinant protein can be subjected to furtherpurification before and/or after the tentacle anion exchange matrixchromatography. Such additional purification steps include but are notlimited to affinity chromatography, ion exchange chromatography,hydroxyapatite chromatography, hydrophobic interaction chromatography, agel filtration (size exclusion) chromatography, mixed modechromatography, and/or filtration. In addition, in all of the aboveaspects of the invention, the recombinant protein can also besubsequently formulated into a pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the compiled results of screening and optimizationexperiments for each of the indicated resins as taken from workingexamples 1 through 8. The recovery of the test recombinant protein,etanercept, is graphed on the Y-axis as a function of percent leachedprotein A reduction, which is plotted on the X-axis. Resins used areindicated and were as follows: DEAE Sepharose Fast Flow (GE HealthcareLife Sciences); Eshmuno® Q (EMD Millipore); Fractogel® SO3 (MerckMillipore); HIC (Toyopearl 650-M; ToyoScreen Ether-650M; ToyoScreenPhenyl-650M; ToyoScreen PPG-600M, all from Tosoh Bioscience GmbH); CHTCeramic Hydroxyapatite Type II 80 μm particle resin (BioRadLaboratories, Inc., Hercules, Calif.); Q Sepharose Fast Flow (GEHealthcare Life Sciences); and Fractogel® EMD TMAE HiCap (EMDMillipore).

FIG. 2 is an overlay of the Recovery/Leached Protein A Reduction curvesgraphed in FIG. 1. Symbols used in the graph for each resin areindicated in the figure legend.

DETAILED DESCRIPTION Definitions

Adsorbent: An adsorbent is at least one molecule affixed to a solidsupport or at least one molecule that is, itself, a solid, which is usedto perform chromatography.

Affinity chromatography: Affinity chromatography is chromatography thatutilizes the specific, reversible interactions between biomolecules, forexample, the ability of Protein A to bind to an Fc portion of an IgGantibody, rather than the general properties of a molecule, such asisoelectric point, hydrophobicity, or size, to effect chromatographicseparation. In practice, affinity chromatography involves using anadsorbent, such as Protein A affixed to a solid support, tochromatographically separate molecules that bind more or less tightly tothe adsorbent. See Ostrove (1990) in Guide to Protein Purification,Methods in Enzymology 182: 357-379, which is incorporated herein in itsentirety.

Chromatography: Chromatography is the separation of chemically differentmolecules in a mixture from one another by percolation of the mixturethrough an adsorbent, which adsorbs or retains different molecules moreor less strongly. Molecules that are least strongly adsorbed to orretained by the adsorbent are released from the adsorbent underconditions where those more strongly adsorbed or retained are not.

Contaminant: A contaminant is any foreign or objectionable molecule,particularly a biological macromolecule such as a DNA, an RNA, or aprotein, other than the protein being purified that is present in asample of a protein being purified. Contaminants include, for example,other proteins from cells that secrete the protein being purified andproteins, such as Protein A, that are part of an adsorbent used foraffinity chromatography that may leach into a sample during affinitychromatography.

Purify: To purify a protein means to reduce the amounts of foreign orobjectionable elements, especially biological macromolecules such asproteins or DNA that may be present in a sample of the protein. Thepresence of foreign proteins may be assayed by any appropriate methodincluding gel electrophoresis and staining and/or ELISA assay. Thepresence of DNA may be assayed by any appropriate method including gelelectrophoresis and staining and/or assays employing polymerase chainreaction.

Separate: A protein is separated from a second protein in a mixturecomprising both proteins when the mixture is subjected to a process suchthat at least the majority of the molecules of the protein are removedfrom that portion of the mixture that comprises at least the majority ofthe molecules of the second protein.

Substantially similar: For purposes of the invention, proteins aresubstantially similar if they are at least 80%, preferably at least 90%identical to each other in amino acid sequence and maintain or alter ina desirable manner the biological activity of the unaltered protein.Included in amino acids considered identical for the purpose ofdetermining whether proteins are substantially similar are amino acidsthat are conservative substitutions, unlikely to affect biologicalactivity, including the following: Ala for Ser, Val for Ile, Asp forGlu, Thr for Ser, Ala for Gly, Ala for Thr, Ser for Asn, Ala for Val,Ser for Gly, Tyr for Phe, Ala for Pro, Lys for Arg, Asp for Asn, Leu forIle, Leu for Val, Ala for Glu, Asp for Gly, and these changes in thereverse. See e.g. Neurath et al., The Proteins, Academic Press, New York(1979). The percent identity of two amino sequences can be determined byvisual inspection and mathematical calculation, or more preferably, thecomparison is done by comparing sequence information using a computerprogram such as the Genetics Computer Group (GCG; Madison, Wis.)Wisconsin package version 10.0 program, ‘GAP’ (Devereux et al., 1984,Nucl. Acids Res. 12: 387) or other comparable computer programs. Thepreferred default parameters for the ‘GAP’ program includes: (1) theweighted amino acid comparison matrix of Gribskov and Burgess ((1986),Nucl. Acids Res. 14: 6745), as described by Schwartz and Dayhoff, eds.,Atlas of Polypeptide Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358 (1979), or other comparable comparisonmatrices; (2) a penalty of 30 for each gap and an additional penalty of1 for each symbol in each gap for amino acid sequences; (3) no penaltyfor end gaps; and (4) no maximum penalty for long gaps. Other programsused by those skilled in the art of sequence comparison can also beused.

Description of the Methods of the Invention

The invention provides a method of purifying a recombinant protein froma sample containing a second protein that binds to the protein. Themethod entails subjecting the sample to a tentacle anion exchange matrixchromatography medium under conditions whereby the recombinant proteinbinds to the tentacle anion exchange matrix chromatography medium,followed by eluting the recombinant protein bound to the chromatographymedium in an eluant. Using the methods of the invention, the presentinventors were able to recover at least 85% of the recombinant proteinin the eluant while removing at least 75% of the second contaminatingprotein. Indeed, the methods of the invention were such that in manyconditions, one can recover at least 90% of the recombinant protein inthe eluant while at the same time removing at least 80% of the secondcontaminating protein. Such superior results were not possible with anyother anion, cation, or hydrophobic interaction chromatography typicallyused in industrial production. While not wishing to be bound to anyparticular mechanism, it is thought that the tentacle aspect of theanion exchange matrix chromatography medium gives the methods of theinvention unexpectedly better results than that observed with any othertype of anion exchange resin, or indeed most resins.

The term “tentacle anion exchange matrix chromatography medium” refersto an anion exchange matrix implementing tentacle technology typicallyas disclosed in U.S. Pat. Nos. 6,398,962, 6,149,994, 5,866,673, or5,647987. Anion exchange matrices implementing the tentacle technologyare resin particles comprising, usually on their surface, spacers formedby linear polymer chains (tentacles), wherein functional groups havinganion exchange activity are attached to the tentacles. In a furtherembodiment, the polymer chains forming said tentacles are acrylamidepolymers. In some embodiments, the functional group is selected from thegroup consisting of TMAE (Trimethylaminoethyl-), DMAE(Dimethylaminoethyl-), and DEAE (Diethylaminoethyl-). In someembodiments, the functional group is a strong anion exchanger. In aparticular embodiment, the strong anion exchanger functional group isTMAE.

In one aspect, the resin substrate of the tentacle anion exchange matrixchromatography medium is a methacrylate polymeric resin or apolyvinylstyrene polymeric resin. In one embodiment, the methacrylate orpolyvinylstyrene polymer is crosslinked. In the examples describedherein, the resin particles consist of crosslinked methacrylate polymer.Examples of tentacle anion exchange matrices are Fractogel® EMD TMAE,Fractogel® EMD TMAE HiCap, Fractogel® EMD TMAE MedCap(m), andFractoprep® DEAE ion exchangers (EMD Millipore).

In one aspect, the tentacle anion exchange matrix chromatography mediumcomprises (i) resin particles of methacrylate polymer or of vinylpolymer, such as methacrylate polymer, that can be cross-linkedmethacrylate polymer, (ii) acrylamide tentacles, wherein the acrylamidetentacles are attached to the surface of said resin particles, andwherein TMAE (Trimethylaminoethyl-) groups are attached to theacrylamide tentaclesfunctional .

The methods of the invention are particularly useful for when the secondprotein is a leached protein from an affinity chromatography medium overwhich the recombinant protein has been previously initially purified.Thus, in one embodiment, the sample containing the recombinant proteinand the second protein is obtained as a result of a prior affinitychromatography step. The most common affinity chromatography proteinscurrently in use are Protein A, Protein G, and Protein LG, which areused to bind antibodies or other proteins that contain a C_(H)2/C_(H)3region of an antibody such as Fc fusion proteins. However, the secondprotein can be any other protein that binds to the recombinant proteindepending upon the structure of the recombinant protein.

Protein A is a protein originally discovered in the cell wall ofStapphylococcus that binds specifically to an Fc portion of an IgGantibody. For purposes of the invention, “Protein A” is any proteinidentical or substantially similar to Stapphylococcal Protein A,including commercially available and/or recombinant forms of Protein A.For purposes of the invention, the biological activity of Protein A forthe purpose of determining substantial similarity is the capacity tobind to an Fc portion of IgG antibody.

Protein G is a protein originally discovered in the cell wall ofStreptococcus that binds specifically to an Fc portion of an IgGantibody. For purposes of the invention, “Protein G” is any proteinidentical or substantially similar to Streptococcal Protein G, includingcommercially available and/or recombinant forms of Protein G. Forpurposes of the invention, the biological activity of Protein G for thepurpose of determining substantial similarity is the capacity to bind toan Fc portion of an IgG antibody.

Protein LG is a recombinant fusion protein that binds to IgG antibodiescomprising portions of both Protein G (see definition above) and ProteinL. Protein L was originally isolated from the cell wall ofPeptostreptococcus. Protein LG comprises IgG binding domains from bothProtein L and G. Vola et al. (1994) Cell. Biophys. 24-25: 27-36, whichis incorporated herein in its entirety. For purposes of the invention,“Protein LG” is any protein identical or substantially similar toProtein LG, including commercially available and/or recombinant forms ofProtein LG. For purposes of the invention, the biological activity ofProtein LG for the purpose of determining substantial similarity is thecapacity to bind to an IgG antibody.

There are many commercially available protein A chromatography mediathat may be used for affinity purification, including ProSep®controlled-pore glass resins produced by Millipore, and MabSelect™cross-linked agarose resin products produced by GE Healthcare, formerlyAmersham Biosciences. Both Mab Select and ProSep resins have dynamicbinding capacities approaching greater than 20 g/L, linear flowvelocities for producing commercial quantities of antibodies rangingfrom 200 to 600 cm/hr, and pH stabilities from about 2 to about 10. Bothtypes of resin are chemically stable when exposed to urea and otherreducing agents. A type of protein A chromatography medium that is usedin the examples illustrating the invention herein is Mab Select SuRe™Protein A resin. Mab Select SuRe™ Protein A resin uses an alteredrecombinant form of protein A which has been genetically engineered tobe more stable than native protein A. Leached amounts of protein Aderived from a Mab Select SuRe™ Protein A column can also be removedusing the methods of the invention. Still another commercially availableProtein A chromatography medium is Protein A Sepharose FF resin (GEHealthcare Life Sciences). An example of a commercially availableprotein G chromatography medium is Protein G Sepharose 4 Fast Flow (GEHealthcare Life Sciences). The methods of the invention entail usingknown and yet to be developed affinity resins.

The process of the invention can, in some embodiments, also involve atleast two steps. First, the recombinant protein undergoes apre-purification step of affinity chromatography using the secondprotein affixed to a solid support as an adsorbent. Second, tentacleanion exchange matrix chromatography medium is performed underconditions such that the recombinant protein is bound to the tentacleanion exchange matrix chromatography medium. After an optional washstep, the recombinant protein is eluted from the tentacle anion exchangematrix chromatography medium. The entire process of purifying theprotein may include other steps before and/or after each of these steps,as noted below.

Prior to equilibration and chromatography, the tentacle anion exchangematrix chromatography medium may be pre-equilibrated in a chosensolution, e.g. a salt and/or buffer solution. Pre-equilibration servesthe function of displacing a solution used for regenerating and/orstoring the chromatography medium. One of skill in the art will realizethat the composition of the pre-equilibration solution depends on thecomposition of the storage solution and the solution to be used for thesubsequent chromatography. Thus, appropriate pre-equilibration solutionsmay include the same buffer or salt used for performing thechromatography, optionally, at a higher concentration than is used toperform chromatography. Buffers and salts that can be used forchromatography are discussed below.

Before the sample is applied to the column, the tentacle anion exchangematrix chromatography medium can be equilibrated in the buffer or saltthat will be used to chromatograph the protein. As discussed below,chromatography (and loading of the protein to be purified) can occur ina variety of buffers or salts including sodium, potassium, ammonium,magnesium, calcium, chloride, fluoride, acetate, phosphate, and/orcitrate salts and/or Tris, phosphate, citrate, HEPES, MOPS, and IVIESbuffers. Such buffers or salts can have a pH of at least about 5.5. Insome embodiments, equilibration may take place in a solution comprisinga Tris or a sodium phosphate buffer. Optionally, the Tris or sodiumphosphate buffer is at a concentration between about 0.5 millimolar andabout 50 millimolar, more preferably at a concentration between about 15millimolar and 35 millimolar. Preferably, equilibration takes place at apH of at least about 5.5. Equilibration may take place at pH valuesbetween about 6.0 and about 8.6, preferably at pH values between about7.0 and 8.5. In one aspect, the solution comprises a Tris buffer at aconcentration of about 25 millimolar and at a pH of about 8.

Any or all chromatographic steps of the invention can be carried out byany mechanical means. Chromatography may be carried out in a column. Thecolumn may be run with or without pressure and from top to bottom orbottom to top. The direction of the flow of fluid in the column may bereversed during the chromatography process. Chromatography may also becarried out using a batch process in which the solid support isseparated from the liquid used to load, wash, and elute the sample byany suitable means, including gravity, centrifugation, or filtration.Chromatography may also be carried out by contacting the sample with afilter that adsorbs or retains some molecules in the sample morestrongly than others.

Conditions for binding the recombinant protein to the tentacle anionexchange matrix chromatography medium can be determined by one of skillin the art and are dependent upon the charge of the recombinant proteinand the strength of the anion functional group. Optionally, after therecombinant protein is bound to the tentacle anion exchange matrixchromatography medium and before the recombinant protein is eluted, thetentacle anion exchange matrix chromatography medium can be subjected toa wash step. The wash buffer is typically the same buffer system inwhich the sample has been prepared and subjected to the tentacle anionexchange matrix chromatography medium, although one of skill in the artwill be able to determine other buffer conditions for washing thechromatography resin without eluting the recombinant protein. If a washstep is included, the volume of the wash can be small or can be severalcolumn volumes. However, typically, the column is washed with from 0.5to 10 column volumes, more typically from 1 to 5 column volumes. Afterbinding of the recombinant protein to the tentacle anion exchange matrixchromatography medium, and optionally washing the medium, therecombinant protein is eluted by increasing the conductivity and/orreducing the pH of the buffer used to chromatograph the sample. Thebuffer condition that can selectively elute the recombinant protein willdepend, in part, upon the charge of the recombinant protein and thestrength of the anionic group.

In one embodiment, the recombinant protein is the recombinant fusionprotein etanercept (CAS registry no. 185243-69-0). Etanercept is arecombinant fusion of the soluble extracellular domain of the p75 TNFreceptor to the Fc domain of a human IgG1 that is produced in ChineseHamster Ovary (CHO) cells, and is commercially available from Amgen Inc.(Thousand Oaks, Calif.) under the tradename Enbrel®. The invention isillustrated by way of working examples below using etanercept as therecombinant protein. In one embodiment of the invention, a samplecontaining etanercept that has been affinity purified using protein Aaffinity chromatography is subjected to tentacle anion exchange matrixchromatography medium in a buffer at about pH 8. Such a buffer can beany species that buffers well in this pH range such as a phosphatecompound or a Tris(hydroxymethyl)aminomethane (Tris) that has beentitrated to have about pH 8. A suitable Tris buffer is from 20 to 30 mMTris:HCl, more preferably about 25 mM Tris:HCl. Optionally, beforeetanercept is eluted from the tentacle anion exchange matrixchromatography medium, the medium can be washed with the same buffer ora slightly different buffer, as long as etanercept remains bound to themedium. In one aspect, the tentacle anion exchange matrix chromatographymedium is washed with one of the above suitable Tris buffers. In oneaspect, the buffered solution to wash the column consists essentially of25 mM Tris at about pH 8. The column can be washed with from 0.5 to 5column volumes, more typically with from 1 to 3 column volumes.

Following binding of etanercept to the tentacle anion exchange matrixchromatography medium, and optionally washing, it is eluted. Anyconditions that will selectively elute the recombinant proteinetanercept can be used. In one aspect, etanercept is eluted in anelution buffer at a pH of from about 6.5 to about 8.5, more preferablyfrom about 7.2 to about 7.6. Again, any buffer species that buffers wellin this pH range can be used, including but not limited to Tris andphosphate compounds, as well as 3-(N-morpholino)propanesulfonic acid(MOPS), 2-(N-morpholino)ethanesulfonic acid (MES),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), andcitrate buffers. For example, the elution buffer can be from about 10 mMto about 50 mM Tris HCL, and about pH 7 to about pH 8. In one aspect,the elution buffer is about 25 mM Tris HCl, pH 7.2 to pH 7.6. Oneembodiment of an elution buffer is 25 mM Tris HCl, pH 7.2. In addition,the elution buffer can have a salt, such as NaCl and/or sodium sulfate.The concentration of salt can range from 0 mM to 500 mM, or from 100 to200 mM. Accordingly, another alternative elution buffer is 25 mM TrisHCl, pH 7.5 and NaCl from about 150 mM to about 200 mM.

Using the methods of the invention, the instant inventors have been ableto recover very high yields of the desired recombinant protein whileeliminating most of the contaminating second protein. Thus, in oneaspect, the methods of the invention result in at least 80%, morepreferably 85%, even more preferably 90%, and most preferably 95%recovery of the recombinant protein in the eluant. At the same time, atleast 70%, more preferably 75%, still more preferably 80%, even morepreferably 85% of the second contaminating protein is removed from theeluant, or any combination of the above recoveries and removals can beachieved.

Protein concentration of a sample at any stage of purification can bedetermined by any suitable method. Such methods are well known in theart and include: 1) colorimetric methods such as the Lowry assay, theBradford assay, the Smith assay, and the colloidal gold assay; 2)methods utilizing the UV absorption properties of proteins; and 3)visual estimation based on stained protein bands on gels relying oncomparison with protein standards of known quantity on the same gel. Seee.g. Stoschek (1990), Quantitation of Protein, in Guide to ProteinPurification, Methods in Enzymol. 182: 50-68.

By “purifying” a recombinant protein, is meant reducing the level of anundesired contaminant. In the methods of the invention, the undesiredcontaminant is the second protein. The second protein, a complex of therecombinant protein and the second protein, and/or other proteins thatmay be present in a sample of the recombinant protein being purified,can be monitored by any appropriate means. Preferably, the techniqueshould be sensitive enough to detect contaminants in the range betweenabout 0.5 parts per million (ppm) (calculated as nanograms per milligramof the protein being purified) and 500 ppm. For example, enzyme-linkedimmunosorbent assay (ELISA), a method well known in the art, may be usedto detect contamination of the recombinant protein by the secondprotein. See e.g. Reen (1994), Enzyme-Linked Immunosorbent Assay(ELISA), in Basic Protein and Peptide Protocols, Methods Mol. Biol. 32:461-466, which is incorporated herein by reference in its entirety.Tentacle anion exchange matrix chromatography may reduce contaminationby a second protein at least about twofold, preferably at least aboutthreefold, more preferably at least about fivefold, still morepreferably at least about tenfold, even more preferably at least aboutfifteenfold, most preferably at least about twentyfold. Preferably,contamination of the recombinant protein by the second protein aftertentacle anion exchange matrix chromatography is not more than about 100ppm, more preferably not more than about 80 ppm, more preferably notmore than about 60 ppm, more preferably not more than about 40 ppm, morepreferably not more than about 20 ppm, more preferably not more thanabout 10 ppm, more preferably not more than about 5 ppm, more preferablynot more than about 1 ppm, and most preferably not more than about 0.5ppm. Contamination by such a second protein can range from undetectablelevels to about 5 ppm or from about 5 ppm to about 400 ppm. If arecombinant protein is being purified for pharmacological use, one ofskill in the art will realize that the preferred level of the secondprotein can depend on the dose of the protein to be administered perpatient, with the aim that the patient will not receive more than acertain amount of a contaminating protein per dose. Thus, if therequired dose of the protein is decreased, the level of contamination bya second protein may possibly increase.

The invention can be used to purify recombinant proteins, which areproteins that have been produced using genetic engineering techniques.Preferably, the proteins are produced at production scale, which istypically in quantities of several grams at a time. The proteinundergoing purification as contemplated by the invention can compriseone or more constant antibody immunoglobulin domain(s) and may, but neednot, comprise a single or multiple variable antibody immunoglobulindomain(s). It may be a naturally-occurring protein or a recombinantfusion protein. It may comprise an Fc portion of an antibody. It mayalso comprise a non-antibody protein.

Examples of recombinant proteins that can be purified with the methodsof the invention include proteins comprising amino acid sequencesidentical to or substantially similar to all or part of one of thefollowing proteins: tumor necrosis factor (TNF), flt3 ligand (WO94/28391), erythropoeitin, thrombopoeitin, calcitonin, IL-2,angiopoietin-2 (Maisonpierre et al. (1997), Science 277(5322): 55-60),ligand for receptor activator of NF-kappa B (RANKL, WO 01/36637), tumornecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, WO97/01633), thymic stroma-derived lymphopoietin, granulocyte colonystimulating factor, granulocyte-macrophage colony stimulating factor(GM-CSF, Australian Patent No. 588819), mast cell growth factor, stemcell growth factor (U.S. Pat. No.6,204,363), epidermal growth factor,keratinocyte growth factor, megakaryote growth and development factor,RANTES, human fibrinogen-like 2 protein (FGL2; NCBI accession no.NM_00682; Rüegg and Pytela (1995), Gene 160:257-62) growth hormone,insulin, insulinotropin, insulin-like growth factors, parathyroidhormone, interferons including α-interferons, γ-interferon, andconsensus interferons (U.S. Pat. Nos. 4,695,623 and 4,897471), nervegrowth factor, brain-derived neurotrophic factor, synaptotagmin-likeproteins (SLP 1-5), neurotrophin-3, glucagon, interleukins, colonystimulating factors, lymphotoxin-β, leukemia inhibitory factor, andoncostatin-M. Descriptions of proteins that can be purified according tothe inventive methods may be found in, for example, Human Cytokines:Handbook for Basic and Clinical Research, all volumes (Aggarwal andGutterman, eds. Blackwell Sciences, Cambridge, Mass., 1998); GrowthFactors: A Practical Approach (McKay and Leigh, eds., Oxford UniversityPress Inc., New York, 1993); and The Cytokine Handbook, Vols. 1 and 2(Thompson and Lotze eds., Academic Press, San Diego, Calif., 2003).

Additionally the methods of the invention would be useful to purifyproteins comprising all or part of the amino acid sequence of a receptorfor any of the above-mentioned proteins, an antagonist to such areceptor or any of the above-mentioned proteins, and/or proteinssubstantially similar to such receptors or antagonists. These receptorsand antagonists include: both forms of tumor necrosis factor receptor(TNFR, referred to as p55 and p75, U.S. Pat. Nos. 5,395,760 and5,610,279), Interleukin-1 (IL-1) receptors (types I and II; EP PatentNo. 0460846, U.S. Patent Nos. 4,968,607, and 5,767,064,), IL-1 receptorantagonists (U.S. Pat. No. 6,337,072), IL-1 antagonists or inhibitors(U.S. Pat. Nos. 5,981,713, 6,096,728, and 5,075,222) IL-2 receptors,IL-4 receptors (EP Patent No. 0 367 566 and U.S. Pat. No. 5,856,296),IL-15 receptors, IL-17 receptors, IL-18 receptors, Fc receptors,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK, WO01/36637 and U.S. Pat. No. 6,271,349), osteoprotegerin (US. Pat. No.6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3, and4), and receptors that comprise death domains, such as Fas orApoptosis-Inducing Receptor (AIR).

Enzymatically active proteins or their ligands can also be purifiedusing the invention. Examples include proteins comprising all or part ofone of the following proteins or their ligands or a proteinsubstantially similar to one of these: a disintegrin andmetalloproteinase domain family members including TNF-alpha ConvertingEnzyme, various kinases, glucocerebrosidase, superoxide dismutase,tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E,apolipoprotein A-I, globins, an IL-2 antagonist, alpha-1 antitrypsin,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

The invention can also be used to purify recombinant fusion proteinscomprising, for example, any of the above-mentioned proteins. Forexample, recombinant fusion proteins comprising one of theabove-mentioned proteins plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fc portion of an immunoglobulin, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; Hakansson et al.(1999), Structure7:255-64. Specifically included among such recombinant fusion proteinsare proteins in which a portion of a protein, including any of the aboveproteins, is fused to an Fc portion of an antibody. Examples of suchproteins are etanercept (a p75 TNFR:Fc) and belatacept (CTLA4:Fc).

The invention can also be used to purify antibodies or portions thereof.The term “antibody” includes reference to both glycosylated andnon-glycosylated immunoglobulins of any isotype or subclass or to anantigen-binding region thereof that competes with the intact antibodyfor specific binding, unless otherwise specified, including human,humanized, chimeric, multi-specific, monoclonal, polyclonal, andoligomers or antigen binding fragments thereof. Antibodies can be anyclass of immunoglobulin. Also included are proteins having an antigenbinding fragment or region such as Fab, Fab′, F(ab′)₂, Fv, diabodies,Fd, dAb, maxibodies, single chain antibody molecules, complementaritydeterming region (CDR) fragments, scFv, diabodies, triabodies,tetrabodies and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding toa target polypeptide. The term “antibody” is inclusive of, but notlimited to, those that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from a host celltransfected to express the antibody.

Examples of antibodies that can be purified using the methods of theinvention include, but are not limited to, those that recognize any oneor a combination of proteins including, but not limited to, theabove-mentioned proteins and/or the following antigens: CD2, CD3, CD4,CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52,CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β, IL-2, IL-3, IL-7, IL-4,IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-13receptor, IL-18 receptor subunits, FGL2, PDGF-β and analogs thereof (seeU.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF, TGF, TGF-β2, TGF-β1, EGFreceptor (see U.S. Pat. No. 6,235,883) VEGF receptor, hepatocyte growthfactor, osteoprotegerin ligand, interferon gamma, B lymphocytestimulator (BlyS, also known as BAFF, THANK, TALL-1, and zTNF4; see Doand Chen-Kiang (2002), Cytokine Growth Factor Rev. 13(1): 19-25), C5complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen,LCG (which is a gene product that is expressed in association with lungcancer), HER-2, a tumor-associated glycoprotein TAG-72, the SK-1antigen, tumor-associated epitopes that are present in elevated levelsin the sera of patients with colon and/or pancreatic cancer,cancer-associated epitopes or proteins expressed on breast, colon,squamous cell, prostate, pancreatic, lung, and/or kidney cancer cellsand/or on melanoma, glioma, or neuroblastoma cells, the necrotic core ofa tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins,TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α, the adhesionmolecule VAP-1, epithelial cell adhesion molecule (EpCAM), intercellularadhesion molecule-3 (ICAM-3), leukointegrin adhesin, the plateletglycoprotein gp IIb/IIIa, cardiac myosin heavy chain, parathyroidhormone, rNAPc2 (which is an inhibitor of factor VIIa-tissue factor),MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumornecrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-y-1 receptor, HLA-DR 10 beta, HLA-DRantigen, L-selectin, Respiratory Syncitial Virus, human immunodeficiencyvirus (HIV), hepatitis B virus (HBV), Streptococcus mutans, andStaphlycoccus aureus. Specific examples of known antibodies which can beproduced using the methods of the invention include but are not limitedto adalimumab, bevacizumab, infliximab, abciximab, alemtuzumab,bapineuzumab, basiliximab, belimumab, briakinumab, canakinumab,certolizumab pegol, cetuximab, conatumumab, denosumab, eculizumab,gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan, labetuzumab,mapatumumab, matuzumab, mepolizumab, motavizumab, muromonab-CD3,natalizumab, nimotuzumab, ofatumumab, omalizumab, oregovomab,palivizumab, panitumumab, pemtumomab, pertuzumab, ranibizumab,rituximab, rovelizumab, tocilizumab, tositumomab, trastuzumab,ustekinumab, zalutumumab, and zanolimumab.

The protein can be produced by living host cells that have beengenetically engineered to produce the protein. Methods of geneticallyengineering cells to produce proteins are well known in the art. Seee.g. Ausubel et al., eds. (1990), Current Protocols in Molecular Biology(Wiley, N.Y.). Such methods include introducing nucleic acids thatencode and allow expression of the protein into living host cells. Thesehost cells can be bacterial cells, fungal cells, or, preferably, animalcells grown in culture. Bacterial host cells include, but are notlimited to, Escherichia coli cells. Examples of suitable E. coli strainsinclude: HB101, DH5α, GM2929, JM109, KW251, NM538, NM539, and any E.coli strain that fails to cleave foreign DNA. Fungal host cells that canbe used include, but are not limited to, Saccharomyces cerevisiae,Pichia pastoris and Aspergillus cells. A few examples of animal celllines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3,and WI38. New animal cell lines can be established using methods wellknow by those skilled in the art (e.g., by transformation, viralinfection, and/or selection). Optionally, the protein can be secreted bythe host cells into the medium.

After the recombinant protein is produced by the cells, it is harvested.If the protein is secreted by the cells into the culture medium, thecells and debris are removed from the culture medium by any of a numberof known techniques such as centrifugation, filtration, and/orflocculation. If the recombinant protein collects inside the cell wallor cell membrane, other known techniques are used to collect and, ifnecessary, solubilize the recombinant protein for subsequentpurification operations.

Typically, affinity chromatography can be used as the first purificationstep. The recombinant protein in solution is bound to an affinitychromatography medium, the medium is washed, and the recombinant proteinis eluted by disrupting the binding of the recombinant protein to theaffinity ligand. This eluted sample containing the recombinant proteinand the affinity ligand as a contaminant or second protein is thensubjected to the methods of the invention on a tentacle anion exchangechromatography medium as described in more detail above.

After the recombinant protein is eluted from the tentacle anion exchangematrix chromatography medium, the eluant containing the recombinantprotein can be subjected to a further purification step. Alternativelyor in addition, the sample containing the recombinant protein can besubjected to an additional purification step before being subjected tothe tentacle anion exchange matrix chromatography in the methods of theinvention. Such further purification steps can be a another ion exchangechromatography step (anion and/or cation), another affinitychromatography step, a metal affinity chromatography step, ahydroxyapatite chromatography step, a hydrophobic interactionchromatography step, a gel filtration (size exclusion) chromatographystep, and/or a mixed mode chromatography resin. Examples of commerciallyavailable resins for such chromatography steps include but are notlimited to CHT Ceramic Hydroxyapatite Type II 80 μm particle resin(BioRad Laboratories, Inc., Hercules, Calif.), DEAE Sepharose Fast Flow(GE Healthcare Life Sciences), Eshmuno® Q (EMD Millipore), Fractogel®SO3 (Merck Millipore), Toyopearl 650-M, ToyoScreen Ether-650M,ToyoScreen Phenyl-650M, ToyoScreen PPG-600M (Tosoh Bioscience GmbH), SPSepharose Fast Flow (GE Healthcare Life Sciences), IMAC Sepharose 6 FastFlow (GE Healthcare Life Sciences), and Q Sepharose Fast Flow (GEHealthcare Life Sciences). In addition, the recombinant protein can befurther purified by filtration. Filtration can be direct (such as blockor cake filtration), or can be tangential flow filtration.

In one aspect, the recombinant protein, such as etanercept, is subjectedto affinity chromatography over a Protein A chromatography medium. Afterthe recombinant protein is eluted, the sample containing the recombinantprotein can be purified over a tentacle anion exchange matrixchromatography medium using the methods of the invention as describeabove. However, before or after subjecting the recombinant protein tothe tentacle anion exchange matrix chromatography medium, an additionalchromatography step can be performed. For example, the recombinantprotein can be subjected to hydroxyapatite chromatography in a flowthrough mode to enhance removal of protein A and other contaminants, oranion or cation exchange chromatography (flow through or bind andelute), or hydrophobic interaction chromatography (flow through or bindand elute).

After the recombinant protein is eluted from the tentacle anion exchangematrix chromatography, the protein can be subjected to furtherpurification steps as noted above before it is formulated, or it can bedirectly formulated. The term “formulated” means the protein is bufferexchanged, sterilized, bulk-packaged, and/or packaged for a final user.In one embodiment, the recombinant protein is formulated in apharmaceutical composition. Suitable formulations for pharmaceuticalcompositions include those described in Remington's PharmaceuticalSciences, 18th ed. 1995, Mack Publishing Company, Easton, Pa.

The following examples are offered by way of illustration and notlimitation.

EXAMPLES Example 1

The goal of these experiments was to effectively remove contaminatingProtein A from a recombinant protein preparation while recovering asmuch recombinant protein as possible. A recombinant protein, etanercept,was expressed in a transformed CHO (Chinese Hamster Ovary) cell culture,and secreted into the medium. After removal of cells from the medium,etanercept was initially purified by running the medium over a MabSelect SuRe™ Protein A resin column (GE Healthcare Life Sciences).Leached protein A was determined using a sandwich ELISA assay.Microtitre plates were coated with a chicken anti-protein A antibody asa capture antibody that was specifically raised against the Mab SelectSuRe ligand. After blocking and washing steps, a biotinylated chickenanti-protein A antibody was used as the detection antibody. The amountof leached protein A in the sample after initial purification over theMab Select SuRe™ Protein A resin column ranged from about 1 ppm to about20 ppm.

In the following experiments, the initially purified etanercept was runover a CHT Ceramic Hydroxyapatite Type II 80 μm particle resin (BioRadLaboratories, Inc., Hercules, Calif.) under the following conditions.The yield of etanercept, expressed as a %, was determined using ELISA onthe pre and post sample collection. The amount of leached protein Areduction, also expressed as a %, was determined before and afterHydroxyapatite chromatography using the sandwich ELISA described above.

Resin: Hydroxyapatite Elut. Leached Load Buffer Protein A Cond. LoadElut. Cond. Yield Red. Equil. Buffer (mS/cm) pH Wash Buffer pH (mS/cm)(%) (%) 25 mM Sodium 3.02 6.69 25 mM Sodium 6.8 2.9 96.8 20.3 Phosphate,pH 6.8 Phosphate, pH 6.8 25 mM Sodium 2.96 6.74 25 mM Sodium 6.83 2.9 9351.4 Phosphate, pH 6.8 Phosphate, pH 6.8 25 mM Sodium 2.98 6.72 25 mMSodium 6.83 2.9 96.4 11.5 Phosphate, pH 6.8 Phosphate, pH 6.8 25 mMSodium 2.98 6.72 25 mM Sodium 6.83 2.9 95.9 16.7 Phosphate, pH 6.8Phosphate, pH 6.8 25 mM Sodium 2.98 6.72 25 mM Sodium 6.83 2.9 96.8 17.9Phosphate, pH 6.8 Phosphate, pH 6.8 25 mM Sodium 2.98 6.72 25 mM Sodium6.83 2.9 93.6 17.9 Phosphate, pH 6.8 Phosphate, pH 6.8 25 mM Sodium 2.986.72 25 mM Sodium 6.83 2.9 95.9 11.5 Phosphate, pH 6.8 Phosphate, pH 6.825 mM Sodium 2.81 6.76 25 mM Sodium 6.72 2.9 94 19.4 Phosphate, pH 6.8Phosphate, pH 6.8 25 mM Sodium 2.81 6.76 25 mM Sodium 6.72 2.9 92 29Phosphate, pH 6.8 Phosphate, pH 6.8 25 mM Sodium 2.81 6.76 25 mM Sodium6.72 2.9 95.2 9.68 Phosphate, pH 6.8 Phosphate, pH 6.8 25 mM Sodium 2.816.76 25 mM Sodium 6.72 2.9 92.7 30.6 Phosphate, pH 6.8 Phosphate, pH 6.825 mM Sodium 2.81 6.76 25 mM Sodium 6.72 2.9 95.3 19.4 Phosphate, pH 6.8Phosphate, pH 6.8 25 mM Sodium 3.16 6.72 25 mM Sodium 6.82 2.9 96.1 40.7Phosphate, pH 6.8 Phosphate, pH 6.8 25 mM Sodium 3.16 6.72 25 mM Sodium6.82 2.9 97.3 39.5 Phosphate, pH 6.8 Phosphate, pH 6.8 100 mM Sodium15.58 6.87 100 mM Sodium 6.84 13.6 92 77.6 Acetate, 100 mM Acetate, 100mM Sodium Chloride, Sodium Chloride, 3M Sodium 3M Sodium Phosphate,Phosphate, pH 6.8 pH 6.8 5 mM Sodium not 6.9 5 mM Sodium 6.89 0.661 93.131.1 Phosphate recorder Phosphate 10 mM Sodium 5.54 6.94 10 mM Sodium6.85 1.25 97.3 12.5 Phosphate Phosphate 50 mM MES, 5.92 6.75 50 mM MES,6.81 2.88 98.7 12.7 5 mM Sodium 5 mM Sodium Phophate Phosphate 100 mMSodium 4.33 6.77 100 mM Sodium 6.75 14.77 93.6 50 Acetate, 3 mM Acetate,3 mM Sodium Phosphate, Sodium Phosphate, 65 mM Sodium 65 mM SodiumChloride, Chloride, pH 6.75, cond pH 6.75, cond 14.77 14.77 100 mMSodium 9.68 6.96 100 mM Sodium 6.73 17.96 80.8 61.8 Chloride, 100 mMChloride, 100 mM Sodium Acetate, Sodium Acetate, 3 mM Sodium 9 mM SodiumPhosphate, pH 6.8 Phosphate, pH 6.8 100 mM Sodium 8.13 7 100 mM Sodium6.8 18.99 86.9 57 Chloride, 100 mM Chloride, 100 mM Sodium Acetate,Sodium Acetate, 3 mM Sodium 12 mM Sodium Phosphate, pH 6.8 Phosphate, pH6.8 100 mM Sodium 7.64 6.9 100 mM Sodium 6.8 18.46 97.1 82.4 Chloride,100 mM Chloride, 100 mM Sodium Acetate, Sodium Acetate, 3 mM Sodium 6 mMSodium Phosphate, pH 6.8 Phosphate, pH 6.8 100 mM Sodium 9.72 6.91 100mM Sodium 6.73 17.96 93.3 63.6 Chloride, 100 mM Chloride, 100 mM SodiumAcetate, Sodium Acetate, 3 mM Sodium 9 mM Sodium Phosphate, pH 6.8Phosphate, pH 6.8 100 mM Sodium not not 100 mM Sodium 6.8 18.46 92.965.5 Chloride, 100 mM recorder recorded Chloride, 100 mM Sodium Acetate,Sodium Acetate, 3 mM Sodium 6 mM Sodium Phosphate, pH 6.8 Phosphate, pH6.8 100 mM Sodium 14.16 6.84 100 mM Sodium 6.8 18.99 88.3 30.3 Chloride,100 mM Chloride, 100 mM Sodium Acetate, Sodium Acetate, 3 mM Sodium 12mM Sodium Phosphate, pH 6.8 Phosphate, pH 6.8 100 mM Sodium 8.07 6.94100 mM Sodium 6.8 18.46 98.8 61.8 Chloride, 100 mM Chloride, 100 mMSodium Acetate, Sodium Acetate, 3 mM Sodium 6 mM Sodium Phosphate, pH6.8 Phosphate, pH 6.8 100 mM Sodium 14.2 6.88 100 mM Sodium 6.8 18.4690.5 23.6 Chloride, 100 mM Chloride, 100 mM Sodium Acetate, SodiumAcetate, 3 mM Sodium 6 mM Sodium Phosphate, pH 6.8 Phosphate, pH 6.8 100mM Sodium 13.7 6.83 100 mM Sodium 6.8 18.99 79.3 58.2 Chloride, 100 mMChloride, 100 mM Sodium Acetate, Sodium Acetate, 3 mM Sodium 12 mMSodium Phosphate, pH 6.8 Phosphate, pH 6.8 100 mM Sodium 7.57 6.9 100 mMSodium 6.8 18.99 91.5 84.8 Chloride, 100 mM Chloride, 100 mM SodiumAcetate, Sodium Acetate, 3 mM Sodium 12 mM Sodium Phosphate, pH 6.8Phosphate, pH 6.8 100 mM Sodium 9.88 6.89 100 mM Sodium 6.73 17.96 88.463 Chloride, 100 mM Chloride, 100 mM Sodium Acetate, Sodium Acetate, 3mM Sodium 9 mM Sodium Phosphate, pH 6.8 Phosphate, pH 6.8 100 mM Sodium8.16 6.92 100 mM Sodium 6.8 18.99 98.7 55.5 Chloride, 100 mM Chloride,100 mM Sodium Acetate, Sodium Acetate, 3 mM Sodium 12 mM SodiumPhosphate, pH 6.8 Phosphate, pH 6.8 100 mM Sodium not not 100 mM Sodium6.8 18.99 88.4 9.09 Chloride, 100 mM recorded recorded Chloride, 100 mMSodium Acetate, Sodium Acetate, 3 mM Sodium 12 mM Sodium Phosphate, pH6.8 Phosphate, pH 6.8 100 mM Sodium 16.15 6.72 100 mM Sodium 6.77 21.198.5 −1.5 Chloride, 100 mM Chloride, 100 mM Sodium Acetate, SodiumAcetate, 25 mM Sodium 25 mM Sodium Phosphate, pH 6.8 Phosphate, pH 6.8100 mM Sodium 15.63 6.76 100 mM Sodium 6.8 21.8 97.9 13.2 Chloride, 100mM Chloride, 100 mM Sodium Acetate, Sodium Acetate, 12 mM Sodium 12 mMSodium Phosphate, pH 6.8 Phosphate, pH 6.8 100 mM Sodium not not 100 mMSodium 6.82 20 99 67.6 Chloride, 100 mM recorded recorded Chloride, 100mM Sodium Acetate, Sodium Acetate, 3 mM Sodium 3 mM Sodium Phosphate, pH6.8 Phosphate, pH 6.8 100 mM Sodium 9.88 6.96 100 mM Sodium 6.77 21.1102 −1.5 Chloride, 100 mM Chloride, 100 mM Sodium Acetate, SodiumAcetate, 25 mM Sodium 25 mM Sodium Phosphate, pH 6.8 Phosphate, pH 6.8100 mM Sodium 8.83 6.88 100 mM Sodium 6.8 21.8 94.6 42.6 Chloride, 100mM Chloride, 100 mM Sodium Acetate, Sodium Acetate, 12 mM Sodium 12 mMSodium Phosphate, pH 6.8 Phosphate, pH 6.8 100 mM Sodium 8.24 6.86 100mM Sodium 6.82 20 87.4 82.4 Chloride, 100 mM Chloride, 100 mM SodiumAcetate, Sodium Acetate, 3 mM Sodium 3 mM Sodium Phosphate, pH 6.8Phosphate, pH 6.8

Using hydroxyapatite chromatography in a flow through mode, it waspossible to remove over 80% of the leached protein A while recoveringover 90% of the etanercept protein.

Example 2

Using the relatively weak anion exchange resin DEAE Sepharose Fast Flow(GE Healthcare Life Sciences), a variety of different buffers andelution conditions was explored as detailed in the below table.Etanercept and protein A concentrations were determined as in Example 1.

Test Resin: DEAE Sepharose FF Mode/ Elution Leached Equilibration LoadBuffer Protein A and Wash Cond. Load Elution Conductivity Elution YieldReduction Buffer (mS/cm) pH Buffer (mS/cm) Flow Rate (%) (%) Bind/Elute/5.2 8 25 mM Tris, 17.14 0.5 mL/min 80 27.9 25 mM Tris, 150 mM pH 8 NaCl,pH 7.4 Bind/Elute/ 5.2 8 25 mM Tris, 17.14 0.5 mL/min 85 26.2 25 mMTris, 150 mM pH 8 NaCl, pH 7.4 Bind/Elute/ 5.2 8 25 mM Tris, 20 0.5mL/min 85 15.1 25 mM Tris, 175 mM pH 8 NaCl, pH 7.5 Bind/Elute/ 5.2 8 25mM Tris, 20 0.5 mL/min 84 46.3 25 mM Tris, 175 mM pH 8 NaCl, pH 7.5Bind/Elute/ 5.2 8 25 mM Tris, 13.7 0.5 mL/min 86 55.9 25 mM Tris, 125 mMpH 8 NaCl, pH 7.5 Bind/Elute/ 5.2 8 25 mM Tris, 13.7 0.5 mL/min 85 61.325 mM Tris, 125 mM pH 8 NaCl, pH 7.5 Bind/Elute/ 5.2 8 25 mM Tris, 13.30.5 mL/min 72.4 96.3 25 mM Tris, 100 mM pH 8 NaCl, pH 7.4 Bind/Elute/5.2 8 25 mM Tris, 13.3 0.5 mL/min 74.7 93.2 25 mM Tris, 100 mM pH 8NaCl, pH 7.4 Bind/Elute/ 5.2 7.94 25 mM Tris, 29.2 100 cm/hr 94.1 1.7525 mM Tris, 250 mM pH 8 NaCl, pH 7.4 Bind/Elute/ 5.2 7.94 25 mM Tris,13.6 100 cm/hr 77.4 94.6 25 mM Tris, 100 mM pH 8 NaCl, pH 7.4Bind/Elute/ 5.2 7.94 25 mM Tris, 13.6 100 cm/hr 79.8 94.5 25 mM Tris,100 mM pH 8 NaCl, pH 7.4 FlowThrough/ 6.7 11.28 Not not Not 95 25 55 mMapplicable recorded applicable Sodium Phosphate Bind/Elute/ 5 7.4 25 mM12.14 25 cm/hr 64.6 88.2 25 mM Tris Tris, 100 mM pH 7.4 NaCl pH 7.4Bind/Elute/ 5 7.4 25 mM 16.7 25 cm/hr 87.1 9.8 25 mM Tris Tris, 150 mMpH 7.4 NaCl pH 7.4 Bind/Elute/ 5 7.4 25 mM 21.2 25 cm/hr 87.3 −2 25 mMTris Tris, 200 mM pH 7.4 NaCl pH 7.4 Bind/Elute/ 4 7.6 25 mM 12.14 25cm/hr 72.6 94.1 25 mM Tris Tris, 100 mM pH 7.4 NaCl pH 7.4 Bind/Elute/ 47.6 25 mM 16.7 25 cm/hr 89.5 23.5 25 mM Tris Tris, 150 mM pH 7.4 NaCl pH7.4 Bind/Elute/ 4 7.6 25 mM 21.2 25 cm/hr 88.2 11.8 25 mM Tris Tris, 200mM pH 7.4 NaCl pH 7.4 Bind/Elute/ 5 7.4 25 mM 17.98 25 cm/hr 87 11.3 25mM Tris Tris, 165 mM pH 7.4 NaCl pH 7.4 Bind/Elute/ 5 7.4 25 mM 17.98 25cm/hr 84.2 9.43 25 mM Tris Tris, 165 mM pH 7.4 NaCl pH 7.4

As can be seen from the data above, DEAE Sepharose can remove a highpercentage of leached Protein A under certain conditions. However, underthose conditions the recovery of the recombinant protein etanercept wasimpacted.

Example 3

In this experiment, a different anion exchange resin was tested for itsability to remove protein A while maintaining a high recovery rate. Theresin was Eshmuno® Q, which is available from EMD Millipore, a divisionof Merck KGaA, Darmstadt, Germany. All runs were in a bind and elutemode.

Resin: Eshmuno Q Leached Equilibration Load Elution Protein A and WashCond. Load Buffer and Yield Reduction Buffer (mS/cm) pH Conductivity (%)(%) 25 mM Tris, 5 8 25 mM Tris, 93.8 55.6 pH 8, cond. pH 7.4, cond. 5mS/cm 21 mS/cm 25 mM Tris, 5 8 25 mM Tris, 94.4 60.8 pH 8, cond. pH 7.4,cond. 5 mS/cm 21 mS/cm 25 mM Tris, 5 8 25 mM Tris, 92 64.7 pH 8, cond.pH 7.4, cond. 5 mS/cm 21 mS/cm 25 mM Tris, 5 8 25 mM Tris, 92.5 63.3 pH8, cond. pH 7.4, cond. 5 mS/cm 21 mS/cm

Although the recovery of etanercept was high using this resin, reductionof Protein A was not sufficient.

Example 4

The strong cation exchanger Fractogel® SO3 (Merck Millipore) was alsotested under a large variety of conditions detailed in the below table.For all conditions, the mode was bind and elute, equilibration and washbuffer was 75 mM Sodium Acetate, and elution was at 150 cm/hr in a 100mM Sodium Acetate buffer at the indicated salt concentration (expressedin mM).

Test Resin: Fractogel SO3 Leached Load Elution Elution Protein A LoadCond. Elution NaCl Buffer Cond. Yield Reduction pH (mS/cm) pH (mM)(mS/cm) (%) (%) 6 6 5 75 15.4 54.7 74 6 6 6 225 28.1 92.8 −26 5.25 5 5.5150 21.5 112.2 3.16 4.5 6 5 75 15.4 77.5 70.7 6 4 6 75 15.24 95.8 40 6 65 75 15.4 61.1 79 4.5 4 5 75 15.4 69.7 65.8 6 4 5 75 15.4 76.4 35.8 6 46 225 28.1 107.7 −12 6 6 6 75 15.24 85.3 73 4.5 6 6 225 28.1 111.9 −17 64 5 75 15.4 69.8 52.6 4.5 4 6 75 15.24 105.1 32.9 4.5 4 6 225 28.1 107.4−41 6 6 6 225 28.1 91.5 −38 5.25 5 5.5 150 21.5 109.2 8.42 4.5 4 5 22528.9 107.3 −13 4.5 6 5 225 28.9 108.6 8.7 6 6 5 225 28.9 89.5 −6 4.5 4 575 15.4 66.3 69.6 4.5 6 5 75 15.4 73.1 76.1 6 4 5 225 28.9 112.4 −1.1 66 6 75 15.24 81.6 79 5.25 5 5.5 150 21.5 109.4 26.3 4.5 6 6 75 15.2497.9 44.6 4.5 6 5 225 28.9 105.6 7.61 4.5 6 6 225 28.1 103.9 3.26 4.5 46 75 15.24 100.9 34.2 6 4 6 225 28.1 106 −5.3 6 4 6 75 15.24 98 41.1 4.56 6 75 15.24 103.8 50 6 6 5 225 28.9 89.5 −16 4.5 4 5 225 28.9 108.51.27 4.5 4 6 225 28.1 110.1 −13 6 4 5 225 28.9 108.1 −5.3 5.25 5 5.5 15021.5 108.1 7.37 4.97 4.43 5.7 85 16 96.1 49 4.97 4.43 5.7 85 16 99.644.4 5.01 4.51 5.7 85 17.98 101.4 27.5 5 4.49 5.7 85 15.82 94.23 50 54.49 5.7 85 15.82 91.82 27.2 5 4.48 5.7 85 15.82 91.56 53.8 4.51 5 5 10617.41 72.54 45.4 5.01 4.51 5.7 85 17.98 97.4 48.8 5.01 4.51 5.7 85 17.9897.6 47.5 5 4.4 5.7 85 17.45 96.2 14.5 5.01 4.47 5.3 170 23.5 95.3 20.25.01 4.47 5.5 135 20.5 95.6 26.9 5.01 4.47 5.3 100 17.31 87 47.7 5.014.47 5.7 100 17.17 91.5 48 5.01 4.47 5.5 135 20.5 94.8 26.9 5.01 4.475.7 170 23.5 97 26.9 5.03 4.56 5.7 85 16.07 84.02 50.4 5.02 4.54 5.7 8516.07 86.1 52 5.1 4.45 5.7 85 14.97 83.9 56.9 5.028 4.67 5.7 85 15.2182.9 43.4 5.07 4.5 5.7 85 14.99 80.8 40.5 5.09 4.5 5.7 85 14.99 86.640.5

Some of the yields were calculated to be over 100% in this experiment.However, this reflects a difference in the procedure and calculation ofthe load, as opposed to a difference in the function of thechromatography step, and for purposes of the run were taken as a 100%yield. Although the yield was overall very high with this resin, removalof leached protein A was insufficient under all conditions tested.

Example 5

In this experiment, several different hydrophobic interactionchromatography resins were examined for their ability to remove leachedprotein A while maintaining a high level of recovery of the recombinantprotein. The columns were equilibrated and washed with Sodium Citrate,pH 3.8 (except for the last HIC-ToyoScreen Phenyl-650 run which was doneat a higher load pH and washed with 75 mM Sodium Citrate, pH 5.5). Eachof the columns was loaded and run in a flow through mode at about 1ml/minute, and washed with about 3 column volumes.

Resin: HIC Leached Load Elution Protein A Resin Cond. Load ElutionBuffer Cond. Yield Reduction Subtype (mS/cm) pH pH Elution buffer(mS/cm) (%) (%) Toyopearl 11.75 3.8 3.8 75 mM Sodium 5.59 46.9 89 650-MCitrate, pH 3.8 ToyoScreen 11.41 3.82 3.8 75 mM Sodium 5.77 62.6 13Ether-650M Citrate, pH 3.8 ToyoScreen 11.22 3.82 3.8 75 mM Sodium 5.78105.2 9.57 Ether-650M Citrate, pH 3.8 ToyoScreen 11.48 3.81 3.9 75 mMSodium 5.78 104.4 11 Phenyl-650M Citrate, pH 3.8 ToyoScreen 15.03 3.763.8 150 mM Sodium 10.19 104.3 6.73 Phenyl-650M Citrate, pH 3.8ToyoScreen 20.7 3.71 3.7 300 mM Sodium 17.4 103.8 4.5 Phenyl-650MCitrate, pH 3.8 ToyoScreen 8.54 3.15 3.1 75 mM Sodium 3.3 85.7 −4.8Phenyl-650M Citrate, pH 3 ToyoScreen 17.78 5.55 5.6 75 mM Sodium 12.76105.3 −1.7 Phenyl-650M Citrate, pH 5.5 ToyoScreen 11.5 3.81 3.9 75 mMSodium 5.78 105.3 9.57 PPG-600M Citrate, pH 3.8

Although it was possible to recover high amounts of the recombinantprotein etanercept, very little reduction of Protein A was observedunder these conditions.

Example 6

The strong anion exchanger, Q Sepharose Fast Flow (GE Healthcare LifeSciences) was also tested under a number of conditions as illustrated inthe below table.

Resin: Q Sepharose FF Leached Equilibration Load Elution Protein A andWash Cond. Load Elution Buffer Buffer Cond. Yield Reduction Buffer(mS/cm) pH and Elution pH (mS/cm) (%) (%) 25 mM Tris, 5.2 8 25 mM Tris,20 81 47.1 pH 8 175 mM NaCl, pH 7.4 25 mM Tris, 5.2 8 25 mM Tris, 20 8033 pH 8 175 mM NaCl, pH 7.4 25 mM Tris, 5.2 8 25 mM Tris, 19.7 78 47.6pH 8 165 mM NaCl, pH 7.4 25 mM Tris, 5.2 8 25 mM Tris, 19.7 77 40.4 pH 8165 mM NaCl, pH 7.4 25 mM Tris, 5.2 8 25 mM Tris, 18.2 79 59.6 pH 8 150mM NaCl, pH 7.4 25 mM Tris, 5.2 8 25 mM Tris, 18.2 74 59.1 pH 8 150 mMNaCl, pH 7.4 25 mM Tris 4 7.4 25 mM Tris, 12.85 44.5 100 pH 7.4 106 mMNaCl pH 7.4 25 mM Tris 4 7.4 25 mM Tris, 17.11 75.1 77.4 pH 7.4 150 mMNaCl pH 7.4 25 mM Tris 4 7.4 25 mM Tris, 18 78.5 56.6 pH 7.4 165 mM NaClpH 7.4 25 mM Tris 4 7.4 25 mM Tris, 17.11 75.5 50.9 pH 7.4 150 mM NaClpH 7.4 25 mM Tris 4 7.4 25 mM Tris, 18 80.2 77.4 pH 7.4 165 mM NaCl pH7.4 25 mM Tris 4 7.4 25 mM Tris, 20.3 90.7 34 pH 7.4 180 mM NaCl pH 7.425 mM Tris 4 7.4 25 mM Tris, 21.8 89.8 22.6 pH 7.4 190 mM NaCl pH 7.4 25mM Tris 4 7.6 25 mM Tris, 18 81.3 64.7 pH 7.4 165 mM NaCl pH 7.4 25 mMTris 4 7.6 25 mM Tris, 21.2 86.6 17.6 pH 7.4 200 mM NaCl pH 7.4 25 mMTris 5 7.4 25 mM Tris, 16.29 80 67.9 pH 7.4 150 mM NaCl pH 7.4 25 mMTris 5 7.4 25 mM Tris, 16.29 80.4 67.9 pH 7.4 150 mM NaCl pH 7.4 25 mMTris 5 7.4 25 mM Tris, 17.98 87.9 39.6 pH 7.4 165 mM NaCl pH 7.4 25 mMTris 5 7.4 25 mM Tris, 17.98 86.6 45.3 pH 7.4 165 mM NaCl pH 7.4 25 mMTris 5 7.4 25 mM Tris, 19.4 88 28.3 pH 7.4 175 mM NaCl pH 7.4 25 mM Tris5 7.4 25 mM Tris, 19.4 88.3 26.4 pH 7.4 175 mM NaCl pH 7.4 25 mM Tris 47.6 25 mM Tris, 17.98 82.7 61.1 pH 7.4 165 mM NaCl pH 7.4 25 mM Tris 6 725 mM Tris, 18.67 85.9 29.8 pH 7 165 mM NaCl pH 7 25 mM 6 6.8 25 mMSodium 18.53 82.8 31.6 Sodium Phosphate, 165 mM Phosphate pH NaCl, pH6.9 6.8 25 mM 6 6.6 25 mM Sodium 18.21 82.2 28.6 Sodium Phosphate, 165mM Phosphate pH NaCl, pH 6.6 6.6

With Q Sepharose FF, both yield and removal of leached protein A was notsufficient under any one condition.

Example 7

A number of other resins were evaluated in initial experiments (notshown), but showed little promise of achieving the desired level ofpurification and recovery. Hence, they were not subjected to theoptimization experiments shown here.

Example 8

In this experiment, Fractogel® EMD TMAE HiCap (EMD Millipore) was testedunder a variety of conditions as detailed in the below table. All of theruns below were done in a bind and elute mode, and washed with 10, 5, or3 column volumes.

Resin: Fractogel ® EMD TMAE HiCap Leach Load Elution Elution Prot AEquil. Wash Cond. Load Buffer Buffer Cond. Yield Reduct Buffer Buffer(mS/cm) pH Elution Buffer pH (mS/cm) (%) (%) 25 mM 25 mM 4.9 8 25 mMTris, 7.4 19.3 83.9 83.8 Tris, pH 8 Tris, pH 8 150 mM NaCl, pH 7.4 25 mM25 mM 4.9 8 25 mM Tris, 7.6 18.6 82.7 72.8 Tris, pH 8 Tris, pH 8 165 mMNaCl, pH 7.6 25 mM 25 mM 4.9 8 25 mM Tris, 7.6 18.6 80.2 71.4 Tris, pH 8Tris, pH 8 165 mM NaCl, pH 7.6 25 mM 25 mM 4.9 8 25 mM Tris, 7.5 19.381.8 66.7 Tris, pH 8 Tris, pH 8 175 mM NaCl, pH 7.5 25 mM 25 mM 4.9 8 25mM Tris, 7.5 19.3 81.8 66.1 Tris, pH 8 Tris, pH 8 175 mM NaCl, pH 7.5 25mM 25 mM 4.9 8 25 mM Tris, 7.5 19.3 83.2 65.7 Tris, pH 8 Tris, pH 8 175mM NaCl, pH 7.5 25 mM 25 mM 4.9 8 25 mM Tris, 7.5 19.3 84.5 66.7 Tris,pH 8 Tris, pH 8 175 mM NaCl, pH 7.5 25 mM 25 mM 4.9 8 25 mM Tris, 7.618.6 84.6 80.6 Tris, pH 8 Tris, pH 8 165 mM NaCl, pH 7.6 25 mM 25 mM 4.98 25 mM Tris, 7.6 18.6 84 73.2 Tris, pH 8 Tris, pH 8 165 mM NaCl, pH 7.625 mM 25 mM 4.9 8 25 mM Tris, 7.46 15.7 88.4 87.1 Tris, pH 8 Tris, pH 8150 mM NaCl, pH 7.4 25 mM 25 mM 4.9 8 25 mM Tris, 7.46 15.7 90.3 87.3Tris, pH 8 Tris, pH 8 150 mM NaCl, pH 7.4 25 mM 25 mM 4.9 8.1 25 mMTris, 7.41 16.68 72.7 83.7 Tris, pH 8 Tris, pH 8 150 mM NaCl, pH 7.4 25mM 25 mM 4.9 8.1 25 mM Tris, 7.41 16.68 72.6 78.6 Tris, pH 8 Tris, pH 8150 mM NaCl, pH 7.4 25 mM 25 mM 5.2 7.9 25 mM Tris, 7.4 29.2 99.2 16.8Tris, pH 8 Tris, pH 8 250 mM NaCl, pH 7.4 25 mM 25 mM 5.2 7.9 25 mMTris, 7.4 29.2 99.7 6.54 Tris, pH 8 Tris, pH 8 250 mM NaCl, pH 7.4 25 mM25 mM 5.2 7.9 25 mM Tris, 7.5 16.72 86.5 89.4 Tris, pH 8 Tris, pH 8 150mM NaCl, pH 7.4 25 mM 25 mM 5.2 7.9 25 mM Tris, 7.5 16.72 88.5 88.8Tris, pH 8 Tris, pH 8 150 mM NaCl, pH 7.4 25 mM 25 mM 6 8.5 25 mM Tris,8 28.5 101.3 8.62 Tris, pH Tris, pH 250 mM NaCl, 8.5, cond. 8.5, cond.pH 8 6 mS/cm 6 mS/cm 25 mM 25 mM 6 7.5 25 mM Tris, 7 28.9 98.2 10.3Tris, pH Tris, pH 250 mM NaCl, 7.5, cond. 7.5, cond. pH 7 6 mS/cm 6mS/cm 25 mM 25 mM 4 7.5 25 mM Tris, 8 13 43.8 100 Tris, pH Tris, pH 100mM NaCl, 7.5, cond. 7.5, cond. pH 8 4 mS/cm 4 mS/cm 25 mM 25 mM 5 8 25mM Tris, 7.5 21.6 98.2 79.3 Tris, pH Tris, pH 175 mM NaCl, 8, cond. 8,cond. pH 7.5 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25 mM Tris, 7.5 21.6 95.878.4 Tris, pH Tris, pH 175 mM NaCl, 8, cond. 8, cond. pH 7.5 5 mS/cm 5mS/cm 25 mM 25 mM 6 8.5 25 mM Tris, 7 13.9 50.5 100 Tris, pH Tris, pH100 mM NaCl, 8.5, cond. 8.5, cond. pH 7 6 mS/cm 6 mS/cm 25 mM 25 mM 48.5 25 mM Tris, 7 13.9 100.2 6.25 Tris, pH Tris, pH 250 mM NaCl, 8.5,cond. 8.5, cond. pH 7 4 mS/cm 4 mS/cm 25 mM 25 mM 6 8.5 25 mM Tris, 728.9 100 11.2 Tris, pH Tris, pH 250 mM NaCl, 8.5, cond. 8.5, cond. pH 76 mS/cm 6 mS/cm 25 mM 25 mM 4 8.5 25 mM Tris, 8 13 46.2 100 Tris, pHTris, pH 100 mM NaCl, 8.5, cond. 8.5, cond. pH 8 4 mS/cm 4 mS/cm 25 mM25 mM 5 8 25 mM Tris, 7.5 21.6 98.1 82 Tris, pH Tris, pH 175 mM NaCl, 8,cond. 8, cond. pH 7.5 5 mS/cm 5 mS/cm 25 mM 25 mM 4 7.5 25 mM Tris, 713.9 56.2 100 Tris, pH Tris, pH 100 mM NaCl, 7.5, cond. 7.5, cond. pH 74 mS/cm 4 mS/cm 25 mM 25 mM 5 8 25 mM Tris, 7.5 21.6 96.6 81.1 Tris, pHTris, pH 175 mM NaCl, 8, cond. 8, cond. pH 7.5 5 mS/cm 5 mS/cm 25 mM 25mM 6 7.5 25 mM Tris, 8 13 32.9 100 Tris, pH Tris, pH 100 mM NaCl, 7.5,cond. 7.5, cond. pH 8 6 mS/cm 6 mS/cm 25 mM 25 mM 4 7.5 25 mM Tris, 8 1342.2 100 Tris, pH Tris, pH 100 mM NaCl, 7.5, cond. 7.5, cond. pH 8 4mS/cm 4 mS/cm 25 mM 25 mM 6 8.5 25 mM Tris, 7 13.9 43.9 81.9 Tris, pHTris, pH 100 mM NaCl, 8.5, cond. 8.5, cond. pH 7 6 mS/cm 6 mS/cm 25 mM25 mM 5 8 25 mM Tris, 7.5 21.6 97 74.8 Tris, pH Tris, pH 175 mM NaCl, 8,cond. 8, cond. pH 7.5 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25 mM Tris, 7.521.6 96.6 82.9 Tris, pH Tris, pH 175 mM NaCl, 8, cond. 8, cond. pH 7.5 5mS/cm 5 mS/cm 25 mM 25 mM 4 8.5 25 mM Tris, 8 28.5 98.5 25 Tris, pHTris, pH 250 mM NaCl, 8.5, cond. 8.5, cond. pH 8 4 mS/cm 4 mS/cm 25 mM25 mM 6 7.5 25 mM Tris, 8 28.5 95.2 25.9 Tris, pH Tris, pH 250 mM NaCl,7.5, cond. 7.5, cond. pH 8 6 mS/cm 6 mS/cm 25 mM 25 mM 4 7.5 25 mM Tris,7 28.9 98.9 5.77 Tris, pH Tris, pH 250 mM NaCl, 7.5, cond. 7.5, cond. pH7 4 mS/cm 4 mS/cm 25 mM 25 mM 4 8.5 25 mM Tris, 7 13.9 55.6 100 Tris, pHTris, pH 100 mM NaCl, 8.5, cond. 8.5, cond. pH 7 4 mS/cm 4 mS/cm 25 mM25 mM 6 7.5 25 mM Tris, 7 13.9 45 100 Tris, pH Tris, pH 100 mM NaCl,7.5, cond. 7.5, cond. pH 7 6 mS/cm 6 mS/cm 25 mM 25 mM 6 8.5 25 mM Tris,7 28.9 100.3 24.1 Tris, pH Tris, pH 250 mM NaCl, 8.5, cond. 8.5, cond.pH 7 6 mS/cm 6 mS/cm 25 mM 25 mM 5 8 25 mM Tris, 7.5 21.6 97.5 84.7Tris, pH Tris, pH 175 mM NaCl, 8, cond. 8, cond. pH 7.5 5 mS/cm 5 mS/cm25 mM 25 mM 6 8.5 25 mM Tris, 8 13 34.3 100 Tris, pH Tris, pH 100 mMNaCl, 8.5, cond. 8.5, cond. pH 8 6 mS/cm 6 mS/cm 25 mM 25 mM 4 7.5 25 mMTris, 8 28.5 98.6 11.5 Tris, pH Tris, pH 250 mM NaCl, 7.5, cond. 7.5,cond. pH 8 4 mS/cm 4 mS/cm 25 mM 25 mM 5 8 25 mM Tris, 7.5 21.6 95.2 82Tris, pH Tris, pH 175 mM NaCl, 8, cond. 8, cond. pH 7.5 5 mS/cm 5 mS/cm25 mM 25 mM 5 8 25 mM Tris, pH 7.2 20 91.6 70.1 Tris, pH Tris, pH 7.2,cond 20 8, cond. 8, cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25 mMTris, pH 7.2 20 90.6 85.1 Tris, pH Tris, pH 7.2, cond 20 8, cond. 8,cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25 mM Tris, pH 7.2 20 9487.4 Tris, pH Tris, pH 7.2, cond 20 8, cond. 8, cond. mS/cm 5 mS/cm 5mS/cm 25 mM 25 mM 5 8 25 mM Tris, pH 7.2 20 91.4 82.9 Tris, pH Tris, pH7.2, cond 20 8, cond. 8, cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25mM Tris, pH 7.2 20 91.7 88 Tris, pH Tris, pH 7.2, cond 20 8, cond. 8,cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25 mM Tris, pH 7.2 20 87.881.9 Tris, pH Tris, pH 7.2, cond 20 8, cond. 8, cond. mS/cm 5 mS/cm 5mS/cm 25 mM 25 mM 5 8 25 mM Tris, pH 7.2 21.2 91.7 61.4 Tris, pH Tris,pH 7.2, cond 20 8, cond. 8, cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 825 mM Tris, pH 7.2 20 95.7 61.7 Tris, pH Tris, pH 7.2, cond 20 8, cond.8, cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25 mM Tris, pH 7.2 2095.7 65 Tris, pH Tris, pH 7.2, cond 20 8, cond. 8, cond. mS/cm 5 mS/cm 5mS/cm 25 mM 25 mM 5 8 25 mM Tris, pH 7.2 20 91.6 70 Tris, pH Tris, pH7.2, cond 20 8, cond. 8, cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25mM Tris, pH 7.2 20 94.9 65.9 Tris, pH Tris, pH 7.2, cond 20 8, cond. 8,cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25 mM Tris, pH 7.2 20 92.768.4 Tris, pH Tris, pH 7.2, cond 20 8, cond. 8, cond. mS/cm 5 mS/cm 5mS/cm 25 mM 25 mM 5 8 25 mM Tris, pH 7.2 20 97.9 86.2 Tris, pH Tris, pH7.2, cond 20 8, cond. 8, cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25mM Tris, pH 7.2 20 97.4 61.1 Tris, pH Tris, pH 7.2, cond 20 8, cond. 8,cond. mS/cm 5 mS/cm 5 mS/cm 25 mM 25 mM 5 8 25 mM Tris, 7.2 19.6 96 69.6Tris, Tris, pH 180 mM NaCl, 33 mM 8, cond. pH 7.2 NaCl, pH 5 mS/cm 8,cond. 5 mS/cm 25 mM 25 mM 5 8 25 mM Tris, 7.2 19.6 96.3 72 Tris, Tris,pH 180 mM NaCl, 33 mM 8, cond. pH 7.2 NaCl, pH 5 mS/cm 8, cond. 5 mS/cm25 mM 25 mM 5 8 25 mM Tris, 7.2 20 92.7 67.8 Tris, Tris, pH 160 mM NaCl,33 mM 8, cond. pH 7.2 NaCl, pH 5 mS/cm 8, cond. 5 mS/cm 25 mM 25 mM 4.617.91 25 mM Tris, 7.4 not 82.3 93.9 Tris, Tris, 150 mM NaCl, recorded pH8 45 mM pH 7.4 NaCl, pH 8.0 25 mM 25 mM 4.58 7.87 25 mM Tris, 7.4 not82.7 94.3 Tris, Tris, 150 mM NaCl, recorded pH 8 45 mM pH 7.4 NaCl, pH8.0 25 mM 25 mM 4.75 7.87 25 mM Tris, 7.4 not not 92 Tris, Tris, 150 mMNaCl, recorded recorded pH 8 45 mM pH 7.4 NaCl, pH 8.0

As compared to the other anion, cation, and hydrophobic interactionchromatography resins analyzed, Fractogel® EMD TMAE HiCap wassurprisingly good at removing Protein A while allowing for a very highrecovery of the target protein. The results from all of the resins thatwere thoroughly investigated were graphed in FIG. 1. An overlay of allof the resins into one overall scatterplot is shown in FIG. 2. Thegeneral trend for all resins is of decreasing yield with increasingprotein A removal. However, in the fully optimized portion of the data,there is a “shoulder” that contains only results using eitherHydroxyapaptite or Fractogel® EMD TMAE HiCap resins.

The present invention is not to be limited in scope by the specificembodiments described herein that are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

1-20. (canceled)
 21. A method for purifying an antibody from a samplecontaining the antibody and a second protein that binds to the antibody,comprising subjecting the sample to a tentacle anion exchange matrixchromatography medium under conditions whereby the antibody binds to thetentacle anion exchange matrix chromatography medium, followed byeluting the antibody bound to the chromatography medium in an eluant,whereby at least 85% of the antibody is recovered in the eluant and atleast 75% of the second protein is removed from the eluant, wherein thesecond protein is Protein A or Protein G.
 22. The method of claim 21,wherein the antibody is a humanized antibody or human antibody.
 23. Themethod of claim 21, wherein the antibody is adalimumab, bevacizumab,infliximab, abciximab, alemtuzumab, bapineuzumab, basiliximab,belimumab, briakinumab, canakinumab, certolizumab pegol, cetuximab,conatumumab, denosumab, eculizumab, gemtuzumab ozogamicin, golimumab,ibritumomab tiuxetan, labetuzumab, mapatumumab, matuzumab, mepolizumab,motavizumab, muromonab-CD3, natalizumab, nimotuzumab, ofatumumab,omalizumab, oregovomab, palivizumab, panitumumab, pemtumomab,pertuzumab, ranibizumab, rituximab, rovelizumab, tocilizumab,tositumomab, trastuzumab, ustekinumab, zalutumumab, or zanolimumab. 24.The method of claim 21, wherein the antibody is denosumab.
 25. Themethod of claim 21, wherein the tentacle anion exchange matrixchromatography medium comprises a strong anion functional group.
 26. Themethod of claim 25, wherein the strong anion functional group istrimethyl-ammoniumethyl (TMAE).
 27. The method of claim 21, wherein theresin substrate of the tentacle anion exchange matrix chromatographymedium is a methacrylate polymeric resin or a polyvinylstyrene polymericresin.
 28. The method of claim 21, wherein the chromatography medium isa methacrylate polymeric resin.
 29. The method of claim 21, wherein thesample is obtained from affinity purification of the antibody over aProtein A chromatography medium.
 30. The method of claim 21, wherein thesample is subjected to the tentacle anion exchange matrix chromatographymedium at about pH
 8. 31. The method of claim 21, wherein after theantibody is bound to the tentacle anion exchange matrix chromatographymedium and before the antibody is eluted, the tentacle anion exchangematrix chromatography medium is subjected to a wash step.
 32. The methodof claim 31, wherein the wash step comprises washing the tentacle anionexchange matrix chromatography medium with a buffered solution at aboutpH
 8. 33. The method of claim 32, wherein the buffered solution consistsessentially of 25 mM Tris(hydroxymethyl)aminomethane (Tris) at about pH8.
 34. The method of claim 21, wherein the antibody is eluted from thetentacle anion exchange matrix chromatography medium in an elutionbuffer at a pH of from about 7.2 to about 7.5.
 35. The method of claim34, wherein the elution buffer is 25 mM Tris HCl, pH 7.2.
 36. The methodof claim 34, wherein the elution buffer is 25 mM Tris HCl, pH 7.5 andNaCl from about 150 mM to about 200 mM.
 37. The method of claim 21,wherein the antibody is subjected to a further purification step beforeor after the tentacle anion exchange matrix chromatography.
 38. Themethod of claim 37, wherein the antibody is formulated in apharmaceutical composition after the purification.
 39. A method forpurifying an antibody from a sample containing the antibody and a secondprotein that binds to the antibody, comprising subjecting the sample toa tentacle anion exchange matrix chromatography medium in a buffer atabout pH 8, followed by eluting the antibody bound to the chromatographymedium in an eluant, wherein the second protein is Protein A or ProteinG.
 40. The method of claim 39, wherein the antibody is adalimumab,bevacizumab, infliximab, abciximab, alemtuzumab, bapineuzumab,basiliximab, belimumab, briakinumab, canakinumab, certolizumab pegol,cetuximab, conatumumab, denosumab, eculizumab, gemtuzumab ozogamicin,golimumab, ibritumomab tiuxetan, labetuzumab, mapatumumab, matuzumab,mepolizumab, motavizumab, muromonab-CD3, natalizumab,